Mycotoxin is the secondary metabolites secreted by mold or other fungi. The growth of mold and the production of the mycotoxin may occur in various processes including the food's maturity, transportation, processing and storage. Feeds contaminated by mycotoxin can cause animal poisoning and affect their immune systems, and even cause serious public health problems. Mycotoxin pollution has been one of the key factors limiting the development of feed and breeding industry. A quarter of crops are contaminated by mycotoxin in varying degrees in the USA and Canada every year, giving rise to a loss of 50 hundred million dollars in feed and breeding industry. The direct losses caused by mycotoxin in China reaches 100 hundred million RMB every year. Even worse situation occurs in south China due to the humid climate.
In order to get rid of the damage to breeding industry, various physical and chemical methods have been developed to absorb or degrade the mycotoxin in feed. Currently, the most popular methods are to remove the mycotoxin by absorption. However, the absorbent is often not selective, resulting in the loss of other nutrients. In addition, mycotoxin excreted from the body can cause secondary pollution. In contrast, the biological detoxification, using enzymes to specifically degrade mycotoxin in mild conditions, with no involvement of hazard chemicals and no loss of nutrients, is considered to be the best method. Developing high efficiency mycotoxin degrading enzymes is the most important way to solve the problems of mycotoxin pollution and to recover the great loss in feed and breeding industry.
Zearalenone (ZEN) is a kind of estrogen-like toxin produced by Fusarium specie, possessing the resorcylic acid lactone structure (as shown in FIG. 1). ZEN is one of the three most seriously spread mycotoxin, identified firstly in moldy corn by Baldwin (Caldwell, R. W., Tuite, J., Stob, M., Baldwin, R., Zearalenone production by Fusarium species. Applied Microbiology, 1970, 20 (1), 31-4). ZEN is mainly existed in corn, wheat, barley, and millet, etc, and can cause the estrogen disordering such as precocity and reproductive cycle disordering, bringing huge losses to crop farming and breeding industry. In addition, ZEN also has a strong carcinogenicity and can cause breast cancer and esophageal cancer. There are six common natural derivatives of ZEN, of which zearalenol (ZOL, as shown in FIG. 1) is often coexisted with ZEN. ZOL has two isomers, α-ZOL and β-ZOL (FIG. 1 shows a generic representation of ZOL comprising α-ZOL and β-ZOL), of which α-ZOL is the major form with a 30-fold higher toxicity than ZEN.
Naoko Takahashi-Ando (Takahashi-Ando, N., Kimura, M., Kakeya, H., Osada, H., Yamaguchi, I., A novel lactonohydrolase responsible for the detoxification of zearalenone: enzyme purification and gene cloning. The Biochemical Journal 2002, 365 (Pt 1), 1-6) identified a lactone hydrolase ZHD101 from Gliocladium roseum, which becomes the most extensively studied ZEN degradation enzyme currently. ZHD101 hydrolyzes the lactone bond in the resorcylic acid lactone structure, opening the ring structure into a straight chain structure (as shown in FIG. 2), which further spontaneously decarboxylized and isomerized to form the final product. The hydrolysis product cannot combine with estrogen receptors, thereby eliminating toxicity. The gene of ZHD101 has been successfully expressed in heterologous hosts. However, it was found that the α-ZOL was still remained in the solution after ZEN had been completely hydrolyzed, due to the lower activity of ZHD101 towards α-ZOL. Since α-ZOL has an even stronger toxicity than ZEN, a huge amount of ZHD101 is needed to remove α-ZOL and to achieve the complete detoxification. Therefore, it is of great value to improve the degradation activity of ZHD101 towards high toxic α-ZOL.
Therefore, in order to improve the efficiency of detoxification of zearalenone and the derivatives, the present invention intends to increase the activity of ZHD101 towards α-ZOL by genetic modification, while maintaining the activity towards ZEN.